The present invention relates to the field of polysaccharides, in particular to a polysaccharide of Echinacea angustifolia and to a process for the preparation thereof. The polysaccharide can be used in the treatment of pathological conditions in which it is desirable to strengthen the immune defenses.
Echinacea is a plant which originates from North America and Mexico; its therapeutical properties were well known to native Americans, who used it for healing wounds. Due to the fact that Echinacea was deemed able to increase the resistance to infections, during the first years of the last century its use in the treatment of local and generalised infections became widespread. Echinacea, in particular Echinacea angustifolia, is nowadays highly recommended for the treatment of influenza syndromes and in particular for the treatment of cold, for healing wounds and for the treatment of mycosis.
The general action is apparently due to the aspecific stimulation of the immune system and to the sensitisation of germs and pathogens to chemotherapeutics and antibiotics. The cicatrizing properties seem ascribable to the capacity of stabilizing hyaluronic acids through of hyaluronidase inhibition exerted by one of the active principles contained in the plant, i.e. echinacoside, and to the massive macrophages activation induced by polysaccharides. In this way any foci of infection remain localised and accumulation of mucopolysaccharides and hystoplastic material necessary for reparative processes is favoured.
As a large fraction of Echinacea polysaccharides consists of inulin, which cannot be deemed responsible for the above cited properties, there is the need to identify the active polysaccharide and to provide an effective process for the extraction and purification thereof.
Object of the present invention is a polysaccharide from Echinacea angustifolia roots (hereinafter referred to as xe2x80x9cthe polysaccharidexe2x80x9d), having molecular weight of 1.3xc3x97105 Da and consisting of rhamnose, arabinose, galactose and galacturonic acid in 0.5:2.5:1.75:10.25 ratio. In the skeleton of the polysaccharide, straight and branched portions alternate, the straight portions consisting of partially acetylated (9%) and methylated (35%) galacturonic acid residues linked via xcex1-(1-4) bond and the branched portions consisting of an alternation of galacturonic acid and rhamnose, to which side chains containing arabinose and galactose in 2.5:1.75 ratio are attached. The polysaccharide is characterized by 1H-NMR, 13C-NMR spectra and GCP profile as reported in FIGS. 1-3.
The polysaccharide is recovered from roots of spontaneous or cultivated Echinacea angustifolia by means of a process comprising the following steps:
a) removing non-polysaccharide components from the roots by extraction with solvent;
b) extracting the polysaccharide fraction from the roots as directly obtained from the preceding step;
c) isolating the polysaccharide by chromatography of the polysaccharide fraction.
The purpose of step a) is to remove the non-polysaccharide components of the roots, mainly echinacoside and analogues thereof, as well as the large group of alkylamides which are also characteristic components of Echinacea angustifolia (R. Bauer et al., Phytochemistry 28, 505, 1989; Planta Med. 55, 367, 1989). Accordingly, the roots are extracted with a solvent selected from acetone or an alcohol from one to three carbon atoms, optionally in admixture with water, at a temperature ranging from 20xc2x0 C. to the boiling temperature, preferably under reflux. The water content of the solvent must not exceed 40% (v/v). Ethanol at a concentration between 80 and 95% (v/v) is the preferred solvent.
The purpose of step b) is to extract the mixture of the polysaccharide components from the roots. The extraction is carried out with water, acetone or an alcohol from one to three carbon atoms, in admixture with water, at a temperature ranging from 20xc2x0 C. to the boiling temperature of the solvent, preferably from 40 to 70xc2x0 C. When mixtures of solvents are used, the water content will be 60% or higher, preferably 85% (v/v). According to a preferred embodiment of the invention, the solvent is 15% (v/v) ethanol.
Step c) provides the fractioning of the extract from step b) and the separation of the polysaccharide from other polar components of the extract. This step preferably consists in size-exclusion chromatography or ion exchange chromatography.
In the first case, the polysaccharide is purified based on its molecular dimensions: in fact the polysaccharide has a characteristic mass, which is different from that of all the other components. A suitably cross-linked resin can separate chemical species having different dimensions; processing the extract from step b) through a resin of this type (for example Toyopearl HW-65S and Superdex(copyright)200HR) allows to purify the polysaccharide of the invention.
The purification of the polysaccharide by ion exchange chromatography is based on the acidic character of the polysaccharide, due to the presence of carboxy functions in the galacturonic acid units. Processing the extract from step b) through an anion exchange resin allows to retain only the polysaccharide and any other acidic components (which are present in small amounts) and to remove all the neutral or basic components. The resin is washed with a saline or acidic aqueous solution to recover the molecules entrapped by the resin. The salts or the acids in the purified solution are thereafter removed by means of ultrafiltration dialysis. A sufficiently high cutoff (e.g. 10,000 or 100,000 Da) is selected to remove also the acidic impurities in the starting extract and retained by the anionic resin.
Preferred anion exchange resins are strong ion exchange resins, such as Diaion HPA 25 and Q Sepharose(copyright)Fast Flow.
The polysaccharide purified solution obtained according to one of the cromatographic processes described in step c) is then concentrated and dried under vacuum or freeze-dried. The polysaccharide is an ivory-colour powder.
Step c) can also comprise preliminary purification steps, useful for making the chromatography easier. Even though not indispensable, these steps allow to remove a first aliquot of impurities from the extract of step b) and to reduce the amount of resin.
Preliminary Treatments are Selected From:
c1) concentration of the extract from step b) under reduced pressure and subsequent purification by treatment with a mixture of water and acetone or water and an alcohol from one to three carbon atoms, preferably ethanol. The mixture will contain 50-70%, preferably 66.5%, of alcohol or acetone. According to a preferred embodiment of the invention, the residue resulting from concentration of the extract is dissolved at room temperature with three parts of water and diluted under stirring with 7 volumes of 95% ethanol. The precipitated fraction, which contains the polysaccharide, is collected by filtration, washed with ethanol at a concentration from 50 to 70% and subjected to chromatographic purification.
c2) treatment of the polysaccharide fraction from step b), or of the enriched fraction from step 1c), with water at room temperature. In this way the polysaccharide of the invention, which is highly soluble in water, is separated from other sparingly soluble polysaccharides. The extract resulting from step b) (or the fraction enriched in polysaccharides from step c1)) is suspended in water at room temperature and stirred to promote dissolution. The insoluble residue is separated and the aqueous solution is subjected to chromatography.
c3) enzymatic treatment, useful for hydrolising inulin-like oligosaccharides and polysaccharides, which represent one of the main impurities of the extract from step b). The extract (or one of the partially purified products from steps c1) or c2)) is treated, in aqueous solution, with a catalytic amount of inulinase for 10-24 hours. The enzyme is then heat- or trypsin-inactivated and the carbohydrate fragments formed upon hydrolysis are removed by dialysis (tangential ultrafiltration with cutoff higher than 10,000 Da, preferably 100,000 Da). The retentate thus obtained is then subjected to chromatography.
c4) high cutoff ultrafiltration (cutoff higher than 10,000 Da, preferably 100,000 Da) to remove low molecular weight impurities. In this case, the extract from step b) (or one of the partially purified products from steps c1) or c2)) is dissolved in water, preferably 10 or 20 volumes, and dialysed. The retentate, which contains the polysaccharide of the invention, is then subjected to chromatography.
The polysaccharide of the invention showed immune-stimolating properties in mice, in particular proved able to stimulate T-lymphocytes activation and to counteract the effect of cyclosporin A, thus reducing the mortality due to Candida albicans infection. The polysaccharide of the invention can be therefore used for the preparation of medicaments, food supplements or nutraceutical compositions to be administered in situations in which an increase of the immune system body defenses is desirable.
The polysaccharide can be formulated according to conventional techniques, for example according to those described in Remington""s Pharmaceutical Sciences Handbook, XVII ed. Mack Pub., N.Y., U.S.A.
The present invention is hereinafter illustrated by means of some examples.